Abstract

Stroke is the leading cause of disability in much of the world, with few treatment options available. Following unilateral stroke in rats, inosine, a naturally occurring purine nucleoside, stimulates the growth of projections from the undamaged hemisphere into denervated areas of the spinal cord and improves skilled use of the impaired forelimb. Inosine augments neurons’ intrinsic growth potential by activating Mst3b, a component of the signal-transduction pathway through which trophic factors regulate axon outgrowth. The present study investigated whether inosine would complement the effects of treatments that promote plasticity through other mechanisms. Following unilateral stroke in the rat forelimb motor area, inosine combined with NEP1-40, a Nogo receptor antagonist, doubled the number of axon branches extending from neurons in the intact hemisphere into the denervated side of the spinal cord compared to either treatment alone and restored rats’ level of skilled reaching using the impaired forepaw to preoperative levels. Similar functional improvements were seen when inosine was combined with environmental enrichment (EE). The latter effect was associated with changes in gene expression in layer 5 pyramidal neurons of the undamaged cortex well beyond those seen with inosine or EE alone. Inosine is now in clinical trials for other indications, making it an attractive candidate for the treatment of stroke patients.

One such agent is inosine, a naturally occurring purine nucleoside. Inosine diffuses across the cell membrane and activates Mst3b, a protein kinase involved in the signal transduction pathway through which trophic factors stimulate axon growth (Irwin et al., 2006; Lorber et al., 2009). Following unilateral stroke or traumatic brain injury in rats, inosine enhances the ability of neurons in the undamaged hemisphere to extend axon collaterals into brainstem and spinal cord areas that have lost their normal innervation and improves fine motor control with the impaired limb (Chen et al., 2002; Smith et al., 2007; Zai et al., 2009).

Manipulations that counteract myelin-associated inhibitors of axon growth represent an alternative way to improve outcome after stroke. Antibodies to Nogo-A or agents that block signaling through the Nogo receptor (NgR) promote the formation of novel corticospinal tract (CST) projections after stroke and improve use of the impaired forelimb (Papadopoulos et al., 2002; Lee et al., 2004; Tsai et al., 2007). Because these approaches induce plasticity by counteracting cell-extrinsic factors, we investigated whether inosine, an activator of neurons’ intrinsic growth potential, would augment the structural and behavioral effects of an NgR antagonist after stroke.

Post-stroke recovery can also be enhanced by environmental enrichment (EE: Ohlsson and Johansson, 1995; Johansson and Ohlsson, 1996; Biernaskie and Corbett, 2001; Fischer et al., 2007). Because physical activity is a standard component of post-stroke recovery, we also investigated whether inosine would augment the effects of EE. Our results show that inosine combined either with a Nogo receptor antagonist or EE restores rats’ success in a skilled-reaching task to preoperative levels using the impaired forepaw.

Materials and Methods

Stroke Surgery

All procedures were carried out in accordance with NIH Guidelines and with the approval of Children’s Hospital Animal Care and Use Committee. Adult male Sprague-Dawley rats (275–300g; Charles River Laboratories, Wilmington, MA) were maintained on a 12 h light/dark cycle (lights on at 7:00 AM). Surgery was carried out between 8:30 AM and 3:00 PM, with animals from different experimental groups intermingled. Strokes were centered in the right caudal forelimb motor area and included varying amounts of adjacent cortical areas (Zai et al., 2009). Rats were anesthetized with ketamine (75 mg/kg) and Domitor (medetomidine; 0.5 mg/kg), the skin was incised along the midline, muscles and skin were retracted, and a craniotomy was performed to create a rectangular window that extended medio-laterally from the sagittal sinus to the temporal ridge, and rostro-caudally between Bregma +3.5 mm and −2.5 mm. Rose Bengal, a photosensitive dye, was injected into the femoral vein through a small opening in the inner thigh. A fiber-optic cable connected to a xenon light source was positioned over the craniotomy, focusing light directly on the exposed region of the brain to activate Rose Bengal for 30 minutes. A green filter restricted illumination to ca. 525 nm. Under these conditions, Rose Bengal releases free radical species that damage endothelial cells in the exposed portions of the cortical vasculature, resulting in platelet aggregation and focal ischemia. Alzet minimpumps were tucked between the shoulder blades and infusion needles were secured onto the cranium with a silicon-based glue. Incisions were closed with 3-0 silk sutures and cleaned with betadine and ethanol pads. Animals were kept on a warming pad during and after surgery, and were returned to their cages when fully awake, mobile, and able to thermoregulate (as judged by absence of piloe-rection). For 3 days following surgery, animals received twice-daily subcutaneous injections of Buprenex (Buprenorphine; Reckitt Benkiser Pharmaceuticals Inc.) for pain management. Animals in the first study were randomly assigned to receive continuous infusion of saline (0.9%, Baxter Scientific), inosine (50 mM in saline, Sigma-Aldrich), NEP1-40 (500 μM in 2.5% DMSO/97.5% saline), or inosine + NEP1-40 into the lateral ventricle of the undamaged hemisphere via osmotic minipumps (0.25 μl/h, Alzet model 2004, Durect Corporation). In order to achieve rapid drug build-up in the CSF, animals in all studies received a 25 μl intraventricular bolus of the agent to be infused prior to pump placement. Treatments were assigned randomly prior to surgery. Unless noted otherwise, rats were housed in pairs.

The study examining the interactions of inosine and NEP1-40 included two behavioral experiments, one in which rats survived for 4 weeks after stroke and another in which they survived for 8 weeks. In the 4-week study, rats treated with either NEP1-40 alone (N = 11) or NEP1-40 plus inosine (N = 11) were generated concurrently with animals reported in another study (Zai et al., 2009), but were not themselves described previously. Another group of rats, treated either with inosine alone (N = 7) or saline (N = 7), were generated specifically for this study. Post-hoc analysis revealed that the behavioral and anatomical effects of inosine- and saline were nearly identical between the newly-generated cases and the ones reported on earlier, justifying our combining the newly generated inosine- and saline-treated animals with the previously-generated animals treated with NEP1-40. Animals in the 8-week study treated with NEP1-40, either with or without inosine (N = 11 for each), have not been described earlier but were also generated concurrently with saline- and inosine-treated animals reported in a previous study (Zai et al., 2009). Behavioral testing was carried out during daylight hours, with no systematic bias towards type of treatment.

Environmental Enrichment

Following pre-training and surgery, animals for anatomical and behavioral studies were implanted with minipumps delivering either saline (N = 21) or inosine (N = 20) and housed in pairs in standard cages. Three days later, half the rats in each treatment group were selected at random and transferred to specially designed chambers for environmental enrichment (EE). The effects of brain injury can be exacerbated by physiological activity within the first week (Humm et al., 1999) although delaying exposure to EE by just 1–3 days enables the beneficial effects of EE to manifest themselves (Ohlsson and Johansson, 1995; Risedal et al., 2002; Dahlqvist et al., 2004).

The EE chamber was a plexiglas cube 24″ on a side with fixed ladders and a platform to stand on, along with a set of tunneling tubes, toys, and treats that varied daily. At any given time, the chamber contained 4–8 rats, with enough plastic tunnels for each rat to have its own. Rats had free access to water throughout the study and, with the exception of the day prior to behavioral testing, free access to standard rat chow. For the microarray studies, rats were housed individually for 3 days and then either transferred to the EE chambers or left in isolation for 4 more days before preparing tissue for analysis (see below).

Behavioral Testing

Rats were tested for their ability to reach through a narrow slit, retrieve food pellets (Bio-Serv, Inc., Frenchtown, NJ) from a platform, and bring them successfully to their mouth (Allred and Jones, 2004; Luke et al., 2004). Variants of this task are widely used in the field because of its established reliance on the integrity of the motor cortex and descending cortical pathways, and because the results are readily quantified. In addition, the task mimics an essential feature of motor strokes in humans by evaluating perfomance on an acquired ability that requires precise motor control. In the acquisition phase, rats were maintained for 3 days prior to and during training on a diet restricted to the banana-flavored pellets used for the retrieval task. Rats were trained daily for 30–60 minutes with each paw for 2 weeks or until they attained a baseline performance of 20–30 successful reaches in a 2-minute period. Immediately after reaching this criterion, each animal was tested for the number of pellets it grasped successfully and consumed in two 2-minute trials. The average of these became the baseline to which subsequent scores were normalized. Rats had free access to food and water for at least three days between baseline testing and surgery. Post-operative testing was carried out in 10–20 minute sessions at 7, 14, 21, and 28 days after surgery by an experimenter blind to the animals’ treatment. Performance was evaluated in two 2-minute blocks using each paw. Scores were recorded only if animals were able to perform at or near their pre-operative level with the unimpaired paw. In instances in which this did not occur immediately, we waited until rats were more active to test them again. In the 8-week studies, rats were tested weekly as above for 4 weeks while receiving the various agents, and then for an additional 4 weeks after minipumps and catheters were removed. Data were analyzed using a two-way ANOVA (repeat measures) with Bonferroni’s post-test to compare data sets.

Anterograde Tracing of Crossing Fibers

At the completion of behavioral testing, rats were re-anesthetized, the infusion needle and pump were removed, and a craniotomy was performed over the uninjured sensorimotor cortex (SMC). Biotinylated dextran amine (BDA: Molecular Probes: 10,000 MW, 10% wt/vol solution in sterile saline) was used as an anterograde tracer and was injected stereotaxically at depths of 0.5, 1.0, and 2.0 mm below the cortical surface at 18 standardized points covering most of the caudal forelimb motor area (70 nl per injection; Nanoject, Drummond Scientific, Broo-mall, PA). Stereotaxic coordinates of the injection sites are shown in Suppl. Table 1. Two weeks later, animals were anesthetized and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain and spinal cord were dissected and post-fixed in the same fixative for one hour at room temperature and overnight at 4 °C, then transferred successively to 10% and 30% sucrose solutions (4 °C) over the next few days. Tissue was embedded in OCT Tissue Tek Medium (Sakura Finetek USA Inc.) and frozen on dry ice. Forty-micron free-floating sections were cut in the coronal plane on a Frigo-Jung 8500 cryostat and stained with avidin-biotin complex conjugated to horseradish peroxidase (Vectastain ABC Kit; Vector Laboratories) followed by Vector SG (Vector) as a chromagen. Sections were mounted on slides (Fisher Super Frost PLUS) and lightly counterstained with eosin to distinguish grey and white matter boundaries. Six to ten equally spaced sections spanning a distance of 1.2 mm were examined and quantified for (a) axons within the dorsal funiculus (BDA-labeled axon profiles ≥ 40 μm in length in the transverse plane within the dorsal funiculus on the denervated side of the spinal cord); (b) axons within the gray matter (as above, but within the gray matter on the denervated side), and (c) lengthy axons in the gray matter (as above, but ≥ 200 μm in length in the transverse plane). Average numbers of axons were calculated and numbers were converted to axons per mm length of spinal cord. Bouton-like structures were identified under a 100X oil objective as regions over 2X the thickness of the axon shaft. Counts were performed in a 0.5 mm2 box aligned with Laminae VI/VII, and were not extrapolated to any volumetric totals. Camera lucida drawings were performed using representative spinal cord sections from control and treatment groups. Statistical significance was determined by one-way ANOVA followed by Tukey’s Multiple Comparisons Test to evaluate significance between treatment groups.

Determination of Lesion Severity

Lesion sizes were calculated from serial sections through the telencephalon as described earlier (Zai et al., 2009). Lesions were re-drawn onto standard sections from the rat brain atlas (Paxinos and Watson, 1986) using grey/white matter boundaries and standard structures. Representations of the injury were created from tracings of scanned sections in Adobe Photoshop.

Cortical Regeneration Assay

Primary cortical cultures were established from embryonic day 18 (E18) Sprague-Dawley rat brains. All tissue culture reagents were from Invitrogen unless otherwise stated. Brains were dissected in Hibernate E without calcium (BrainBits LLC) supplemented with B-27, 1 mM sodium pyruvate and 10 μg/ml gentamicin (dissection medium). Reagents and brains were kept on ice except during the initial dissection. Meninges were removed, cortices were dissected and combined in a 10 cm Petri dish in 1 ml of dissection medium. Tissue was diced approximately 100 times into small pieces with a razor blade. Dissection medium (12 ml) containing papain (4.2 mg/ml: Sigma, P4762) and DNase (0.8 mg/ml) were added to the Petri dish, which was then placed on an orbital shaker (Fisher Scientific, 11-671-50Q) in a 37° incubator (without CO2 regulation) and shaken at 30 rpm for 1 hour. The dish was removed from the incubator and the medium was transferred to a 15 ml polystyrene Falcon tube on ice. Chunks of cortex were allowed to settle to the bottom of the tube for 1–2 min. The medium was removed and chunks were resuspended in 1.5 ml of dissection medium plus 10% FBS and triturated 10–15 times with a fire-polished Pasteur pipette. Medium containing dissociated cortices was transferred to another 15 ml tube containing 12.5 ml of dissection medium plus 10% FBS and centrifuged at 600 x g for 6 minutes at 4° C. The supernatant was removed and cells were resuspended in 12 ml of Neurobasal A supplemented with B-27, 1 mM sodium pyruvate, 0.5 mM GlutaMAX-I and 10 g/ml gentamicin (in Neurobasal A plus supplements). Cells were filtered through a 70 m cell strainer into a 50 ml Falcon tube and counted. Cells were removed from ice and diluted to 3.33×105 cells/ml in Neurobasal A plus supplements which had been pre-warmed and pre-equilibrated in a 37° tissue culture incubator (5% CO2). Cells were plated at a density of 1.2×105 cells/cm2 (4×104 cells/well in 120 l) in 96-well plates coated with poly-D-lysine (Becton Dickinson Labware, BD BioCoat). Door openings over the ensuing 21 days were kept to a minimum. At 7 and 14 days in vitro (DIV), cultures were fed by replacing 50% of the medium with fresh, pre-warmed and pre-equilibrated Neurobasal A plus supplements. At 21 DIV, cultures were injured by scraping with a 96-well floating pin tool with FP1-WP custom parylene-coated pins (0.557 mm tip diameter). To guide the movement of the pin tool, a library copier (V&P Scientific, VP 381NW4.5) with vertical alignment holes was used. Aluminum guide-rails were added on the left and right of the library copier (Electronic and Machine Shop, Yale Univ. School of Medicine), to minimize lateral movement of the pin tool. Immediately after the scrape, 50% of the medium was replaced with fresh medium. In some cases, the fresh medium contained extracted bovine CNS myelin (Cafferty et al., 2010) and/or agents to be tested. Cultures were returned to the incubator to regenerate neurites for five days.

At 26 DIV, cultures were fixed by adding 100 μl/well of 4% paraformaldehyde + 20% sucrose in PBS for 20 min, then blocked and permeabilized with 10% normal goat serum + 0.1% Triton X-100 in PBS for 30 min. Cultures were immunostained with an anti-βIII- tubulin antibody (Promega, G7121, 1:1000), followed, after rinsing with PBS, by an Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A11029, 1:1000). Images were acquired with an ImageXpress Micro imaging system (Molecular Devices) using a 10X objective. Neurite regeneration was analyzed using MetaXpress Version 1.7 software. The central 75% of the lesion was analyzed by cropping the image and analyzing neurite growth using an angiotube formation algorithm. The area covered by neurites was measured and all values were normalized to the control. Results were analyzed by 1-way ANOVA and Fisher’s least-significant difference post-hoc test.

Microarray studies

We investigated patterns of gene expression in layer 5 pyramidal cells of the forelimb motor area contralateral to the damaged hemisphere in four groups of rats, each containing 3–6 individual animals. These neurons were selected for study because parts of this population, though not necessarily the individual neurons that were isolated, give rise to the novel projections visualized in the anatomical studies. As before, rats underwent stroke surgery and were implanted with minipumps delivering either saline or inosine into the lateral ventricle of the undamaged hemisphere as described above. All rats used in the microarray study were initially housed individually. After 3 days, half the rats in each treatment group were randomly selected and transferred to the EE boxes described above, while the remainder continued to be housed in isolation. To focus on gene changes associated with the early response to EE, rats were anesthetized and killed by decapitation four days later. Brains were rapidly dissected and cut in the coronal plane to prepare tissue blocks that included the forelimb motor cortex. Blocks were rapidly frozen in OCT Tissue Tek Medium (Sakura Finetek USA Inc.) and stored at −80°C until ready for sectioning on a cryostat at 10 μm. Sections were mounted on slides (Gold Seal RITE-ON glass slides), cooled, and stored at − 80° C until ready to use.

Laser-Capture Microdissection (LCM) was carried out as described (Zai et al., 2009). In brief, sections were thawed, dehydrated under RNAse-free conditions, and ≥ 500 layer 5 pyramidal cells in the forelimb motor area of the undamaged hemisphere were captured individually using the Arcturus VERITAS system and stored in Arcturus extraction buffer (−80° C). We extracted RNA using the Micro-to-Midi TotalRNA Purification System (Invitrogen), then carried out two cycles of RNA amplification (TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0: Epicentre Biotechnologies). Microarray hybridization and analysis was performed essentially as described (Carmichael et al., 2008; Zai et al., 2009). Briefly, amplified RNA was checked for average fragment length (RNA 6000 Nano LabChip Kit, Agilent Technologies), biotinylated, and hybridized (1 μg) on RatRef -12 Expression BeadChip arrays (Illumina), querying the expression of >22,000 RefSeq-curated rat transcripts. Slides were processed and scanned (Illumina BeadStation platform), and raw data was analyzed using Bioconductor packages as described (Gentleman et al., 2004). First-level quality-control analysis was performed using clustering based on variance and by comparing gene expression patterns for individual cases to group means (Pearson correlations: Oldham et al., 2008). To decrease noise, 3 cases that showed a relatively low correlation to their respective group means (r2 < 0.94) were excluded, increasing the r2 values for the remaining cases to ≥ 0.97. The final analysis included n = 4 samples from rats treated with saline reared in isolation, n = 4 treated with inosine and reared in isolation, n = 4 treated with saline and exposed to EE, and n = 3 treated with inosine and exposed to EE. Data were normalized using quantile normalization, and analysis of differential expression was performed using linear model fitting (LIMMA package, Smyth et al., 2005). Differentially expressed genes were classified according to gene ontology using Bioconductor packages and online tools (DAVID, http://david.abcc.ncifcrf.gov/). Pathway analysis was carried out using Ingenuity Pathway Analysis (Ingenuity Systems).

Results

Inosine combined with NEP1-40

The first study tested the hypothesis that inosine and NEP1-40, by acting through complementary mechanisms, will lead to greater anatomical reorganization and stronger improvements in performance when combined than in isolation. Failure to confirm this hypothesis would imply that the two treatments act through a common cellular pathway, or that the effects of either one represent an upper limit on corticospinal tract plasticity and/or functional outcome after stroke. Rats were pre-trained prior to surgery to reach for food pellets at a criterion level using each paw. Under stereotaxic guidance, we then used the photothrombotic method of Markgraf et al. (1993) to create unilateral ischemic damage in the caudal forelimb motor area, and immediately began intracranial delivery of inosine or saline, with or without NEP1-40.

Lesion size and localization

As reported earlier (Zai et al., 2009), infarcts had a diameter of 6–7 mm and were generally restricted to cortical tissue and some underlying white matter. In addition to the forelimb motor area, lesions included varying amounts of adjacent cortex but minimal damage to the underlying basal ganglia (Suppl. Fig. 1a). Our prior study (Zai et al., 2009) and another lab (Shen et al., 2005) have shown that inosine does not alter lesion size after stroke. The present results show that lesion size was likewise unaffected by NEP1-40, with or without inosine (Suppl. Fig. 1b).

Combinatorial treatment enhances CST reorganization

To investigate changes in CST organization, we injected biotinylated dextran amine (BDA) into the uninjured sensorimotor cortex (SMC) at the completion of behavioral testing, waited two weeks for BDA to be transported to the cervical spinal cord, then euthanized rats and prepared the brains for histology. Sections were examined for axon growth and synapse formation from CST axons that arise from neurons on the undamaged side of the brain and cross over into the denervated side of the spinal cord at the cervical level. One-way ANOVA revealed an overall effect of treatment on CST axon branches that project from the intact hemisphere into the denervated ipsilateral dorsal funiculus (P< 0.05), labeled fibers that continue on into the denervated neuropil (P < 0.001), crossed fibers that extend long distances through the neuropil (P < 0.0001), and bouton-like structures that arise from crossed CST axons (P < 0.001). Individual comparisons were done using Tukey’s post-hoc analysis. Inosine and NEP1-40 each induced a 3-fold increase in the number of BDA+ fibers that projected from the undamaged cortex to the denervated, ipsilateral dorsal funiculus compared to controls treated with saline alone (all P values < 0.05: Fig. 1a–d,i), and caused a similar increase in the number of CST axon branches ≥ 40 μm in the denervated cervical gray matter (Fig 1j). Both treatments caused a somewhat larger increase in the number of longer fibers (> 200 μm in the transverse plane) (Fig. 1f,g,k; all P values < 0.05: c.f.Zai et al., 2009). As expected, all of these increases co-varied. In addition to the main part of the CST, some crossed CST fibers course through the lateral funiculus (Casale et al., 1988; Rouiller et al., 1991), while a relatively small number of uncrossed CST fibers run along the ventral midline of the spinal cord (Brosamle and Schwab, 1997). It is possible that some CST axons within these bundles may have contributed to the observed increases in axon growth after treatment.

Crossed CST axons formed bouton-like structures in the gray matter on the denervated side of the spinal cord. These structures appear as local swellings ≥ 2x the width of the axons (Fig. 1e – h), and have been shown to correspond to synaptic boutons at the electron microscopic level (Lagerback et al., 1981; Havton and Kellerth, 1987). Camera lucida tracings illustrate the shape and size of both en passant and terminal boutons (Fig. 1e’ – h’). As expected, the numbers of these swellings were generally proportional to the number of crossed axons. Treatment with either inosine or NEP 1-40 alone nearly tripled the number of bouton-like structures compared to saline (Fig. 1l, P < 0.05); combining the two resulted in nearly five times more boutons than were seen in untreated controls (Fig. 1l; P < 0.05 comparing combined treatment with either treatment alone).

In view of the large effect of the combined treatment on the denervated side of the spinal cord, we investigated whether it might also affect CST organization on the intact side. Because the high density of fibers on the intact side precluded a comprehensive fiber count, we focused on lamina 9, a region in which no CST fibers project ordinarily (Yang and Lemon, 2003). After stroke, this region was nearly devoid of CST input in saline-treated animals, and neither inosine nor NEP1-40 altered this situation. However, combining the two increased the number of fibers entering lamina 9 and overlying ventral motor neurons (Suppl. Fig. 2).

Combined therapy restores skilled reaching to near-normal levels

Functional recovery was evaluated using a reaching test that requires pre-training and involves fine motor control of the forelimbs, forepaws and digits. Animals were trained to retrieve food pellets with either paw prior to surgery and both paws were tested afterwards. Scores were normalized by rats’ preoperative level of performance. As expected, all groups showed a near-complete loss in their ability to retrieve food pellets with the paw contra-lateral to the stroke in the first week. Over the next 3 weeks, saline-treated animals recovered to 32% of their pre-operative level of performance (Fig. 2a). Two-way ANOVA (repeat measures) revealed highly significant overall effects of treatment and time (P < 0.0001 for each). By week 4, inosine-treated rats were performing at 73% of their pre-operative level, compared to 32% in saline-treated controls (Fig. 2a; post-hoc analysis using Bonferroni’s correction, P < 0.05). Animals treated with NEP1-40 likewise showed an overall superiority to saline-treated controls and performed at 68% of their preoperative level by week 4 (Fig. 2a: difference significant at P < 0.05). Combining inosine with NEP 1-40 resulted in appreciably better performance than either treatment alone. From week 2 on, rats treated with inosine plus NEP1-40 scored considerably better than rats treated with either inosine alone (all P < 0.05) or NEP1-40 alone (all P < 0.01). By week 4, rats receiving combined treatment were able to retrieve food pellets with the impaired paw at a normal rate (Fig. 2c,d).

Inosine combined with NEP1-40 restores performance with the impaired paw to pre-operative levels

Using the unimpaired paw, animals in all groups showed a small decline in performance at week 1, perhaps due to difficulty in balancing on the impaired limb, but performed normally from week 2 onwards (Fig. 2b). Thus, the deficits observed using the impaired paw cannot be attributed to differences in motivation or overall behavioral competence. To determine whether the benefits of combined treatment persist, we generated new groups of animals that were treated for 4 weeks after stroke as before but then tested for an additional 4 weeks after treatments ended. The saline- and inosine-treated animals of this study have been described previously (Zai et al., 2009). Two-way ANOVA (repeat measures) revealed significant overall effects of treatment and time (P < 0.0001 for each). Animals treated with NEP1-40, with or without inosine, continued to improve after the treatments ended. Rats treated with inosine plus NEP1-40 scored considerably better than those treated with NEP1-40 alone from week 5 on (all P < 0.05, Bonferroni’s post-hoc test), performing at a level at or above their pre-operative baseline (Fig. 2c). Inosine-treated rats run in parallel to the rats of the present study were previously reported to perform at approximately 80% of their preoperative level at week 8 (Zai et al., 2009), and as shown here, rats treated with inosine + NEP1-40 were superior to these from week 4 on (all P < 0.05). As expected, animals in all groups continued to perform normally with the paw contralateral to the uninjured hemisphere (Fig. 2d). The results of this study confirm the effects of NEP1-40 combined with inosine seen in the 4 week study, and show that the benefits of these treatments persist long after treatment ends. Thus, 4 weeks after suffering extensive cortical damage, rats treated with inosine + NEP1-40 scored as high as normal animals in the skilled reaching task using the impaired forelimb and retained this ability in the absence of further treatment.

Inosine combined with NEP 1-40 enhances neurite outgrowth on myelin

Previous studies have shown that inosine promotes outgrowth from embryonic cortical neurons on a permissive substrate (Irwin et al., 2006) and that NEP1-40 enables other types of postnatal neurons to extend axons on myelin (GrandPre et al., 2002). Although cortical cultures are most readily prepared from embryonic cortex or early postnatal brain, these neurons express little or no Nogo receptor 1 (NgR1) at this stage and are therefore of questionable relevance to adult stroke recovery. However, NgR1 expression increases with time in culture and becomes similar to adult brain levels by three to four weeks in vitro (data not shown). We therefore used cortical cultures maintained for 21 DIV to study the individual and combined effects of inosine and NEP1-40 on neurite regeneration. Mature cultures were scraped with a multi-pin tool in 96-well plates to create axonal injuries. Fibers were then allowed to regenerate into the zone cleared of neurites in the presence or absence of myelin for 5 days (Fig. 3A). As expected, the presence of CNS myelin (100 μg/ml) strongly inhibited regeneration across the scrape zone (Fig. 3B, C). Inosine at either 0.1 or 1 mM showed a trend toward increasing outgrowth over myelin, as did NEP1-40 alone. Importantly, combining the two enabled a significant amount of regenerative growth to occur in the presence of myelin, approaching the level of growth seen in the absence of myelin (Fig. 3B, C: 1-way ANOVA, Fisher’s least-significant difference test). This study enables us to directly visualize the combined effect of inosine and NEP1-40 in overcoming myelin inhibition.

Inosine combined with environmental enrichment (EE)

The next set of studies tested the hypothesis that inosine enhances the effects of rearing animals in an enriched environment. Although inosine and EE each improves functional outcome after stroke, it is not known whether EE alters CST organization nor whether the two treatments combined affect outcome to a greater extent than either one alone. This study was initially intended to be independent from the ones described above and differed in several experimental details. Following pre-training and stroke surgery, rats were either reared in pairs or, beginning 3 days after stroke, maintained in a complex environment with opportunities for climbing, exploring, and social interactions. Half the animals in each condition received intraventricular infusions of inosine as before, whereas the other half received saline. Treatments ended after 4 weeks, but exposure to EE (or to a standard environment, SE) and behavioral testing continued for another 4 weeks. Thus, unlike the prior study that involved two independent experiments with 4- and 8-week survival periods, the effects of EE and the persistence of these effects were evaluated in a single 8-week study.

CST reorganization

As in our prior studies, the distribution of axons originating in the intact forelimb motor area was evaluated by anterograde tracing with BDA after completing the behavioral studies. One-way ANOVA revealed a significant overall effect of treatment (P < 0.0001). In rats maintained in a standard environment (SE), inosine increased the number of CST axon branches that projected from the undamaged hemisphere to the denervated side of the spinal cord 5-fold compared to saline-treated controls (Fig. 4a: P < 0.001, Tukey’s post-hoc correction). This increase is greater than that seen in the previous study (Fig. 1j), reflecting a combination of somewhat more growth in rats treated with inosine in the present series compared to the previous study, and relatively less growth in rats treated with saline. Conceivably, these differences could arise from the longer survival times used here (i.e., 8 weeks vs. 4), but this has not been studied systematically. EE also increased CST sprouting (P < 0.05, Fig. 4a) though to a lesser degree than inosine. Contrary to our expectations, inosine combined with EE produced no more sprouting than inosine alone (Fig. 4a).

Behavioral outcome

As expected from the first set of experiments and our prior studies (Zai et al., 2009), inosine improved animals’ ability to retrieve food pellets with the impaired forepaw. Two-way ANOVA (repeat measures) revealed significant overall effects of treatment and time (P < 0.0001 for each). Controls housed in SE conditions after stroke and treated with saline reached a plateau of ~ 45% of their pre-operative level of performance after 4 weeks (Fig. 4b), comparable to the level seen in saline-treated controls in the 8-week study (Fig. 2c), but somewhat higher than in the 4 week study (Fig. 2a). Inosine, in comparison, enabled rats to attain ≥ 80% of their pre-operative success rate by week 7 (P < 0.05 for inosine vs. saline-treated animals at weeks 5, 7, and 8). EE enabled rats to retrieve food pellets at a level intermediate between that of inosine- and saline-treated rats reared in SE. Animals treated with inosine and exposed to EE performed better than all other groups from the fourth week on and continued to improve after treatment ended (P < 0.05 compared to saline-treated controls exposed to SE from week 2 onwards; P < 0.05 compared to saline-treated animals exposed to EE at weeks 7–8: post-hoc analyses using Bonferroni’s correction). By week 7, inosine-treated animals exposed to EE were able to retrieve food pellets at their pre-operative levels (Fig. 4b). Using the forelimb contralateral to the intact hemisphere, scores remained normal for all groups after the first week (Fig. 4c).

Gene expression

To maximize our chances of detecting an effect of environment on gene expression, rats were reared in isolation for 3 days after stroke and were either maintained in this condition or exposed to EE for the next 4 days. At the end of this period, we isolated layer 5 pyramidal cells of the intact hemisphere by laser-capture microdissection and analyzed gene expression using microarrays. Although animals did not evidence an overt behavioral response to EE after 4 days, we hypothesized that a neuronal response would be taking place at this time point.

Analysis of the microarray results revealed a striking interaction between inosine and EE. For animals reared in isolation for a week, inosine barely affected gene expression. This finding can be visualized by the close correlation between the expression levels of individual genes seen in animals treated with inosine after stroke and reared in SE vs. those treated with saline under the same rearing conditions (R2 = 0.98; Fig. 5a). Using as criteria that changes be significant at P ≤ 0.01, ≥ 1.7-fold in magnitude, and that at least one signal is ≥ 27, inosine altered the expression of only 21 genes relative to saline-treated controls (16 increases and 5 decreases: Suppl. Table 2a). One of the increases associated with inosine was Slpi (secretory leukocyte protease inhibitor), a protein that enhances the ability of axons to grow over inhibitory substrates (Hannila and Filbin, 2008). Two of the 5 decreases were for per genes, which are involved in circadian regulation. Using less stringent criteria (changes significant at P < 0.01, magnitude of the changes unconstrained), gene networks affected by inosine when animals were reared in isolation included “Gene expression, connective tissue development and function and tissue morphology” (14/34 genes), “Cell-to-Cell Signaling and interaction” (14/41 genes), Inflammatory response/cellular movement (13/35 genes) and cellular growth/inflammatory response (6/34 genes). These effects overlapped slightly with canonical pathways involved in regulating circadian rhythm (3/33 genes, P ~ 0.0025, Ingenuity Pathway Analysis, IPA) and CD27 Signaling in Lymphocytes (P ~ 0.01, 3/56 genes).

An alternative way to analyze these results is by examining the effect of varying environmental rearing conditions when rats were treated with inosine or saline. EE had little effect when rats were treated with saline, increasing the expression of only one gene (Gfra2, the neurturin receptor) > 1.7-fold compared to animals reared in isolation, and decreasing the expression of 28 genes by ≤ 1/1.7 (P < 0.01, at least one signal ≥ 128: Suppl. Table 2c). Accordingly, gene expression levels in layer 5 pyramidal cells were highly correlated between rats exposed to EE vs. rats reared in isolation (R2 = 0.97, Fig. 5c). Using the criterion of P < 0.01 without constraining the magnitude of the changes, affected networks included “lipid metabolism, molecular transport and small molecule biochemistry” (13/35 genes), and “developmental disorders, cellular growth and proliferation, and tissue morphology” (10/35 genes). We did not observe effects on a large percentage of genes in any particular canonical pathway.

These results show that inosine and EE interact strongly with one another to alter gene expression after stroke. In rats reared in isolation, inosine barely affected gene expression in layer 5 pyramidal cells of the forelimb motor area contralateral to the stroke. Similarly, when rats were treated with saline, EE had little effect on gene expression. However, in the presence of inosine, EE profoundly altered gene expression. A heat-map of these results (Fig. 5e) shows the intra-group similarities in patterns of gene expression and the strong effect of combinatorial treatment. The molecular changes observed when rats were treated with inosine and exposed to EE mirror the synergistic effects of the two treatments on behavioral outcome, providing a molecular correlate of this phenomena and a foundation for investigating the complex mechanisms involved.

Discussion

Inosine diffuses across the cell membrane and activates the protein kinase Mst3b, a key part of the signal transduction pathway through which growth factors induce neurons to extend axons (Irwin et al., 2006; Lorber et al., 2009). Our results confirm earlier studies showing that inosine exerts a strong effect on axonal reorganization and functional outcome after stroke, while demonstrating for the first time that it enhances the effects of treatments that promote growth through complementary mechanisms. When combined with either a peptide antagonist to the Nogo receptor or environmental enrichment, inosine enabled rats with cortical strokes to reach through a restricted opening and retrieve food pellets with their impaired forelimbs at the same rate as normal animals.

Inosine enhances the effects NEP1-40

Three myelin-associated proteins, Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp), suppress axon growth via NgR (Fournier et al., 2001; Filbin, 2003; Liu et al., 2006) and other receptors (Atwal et al., 2008; Goh et al., 2008; Hu and Strittmatter, 2008). Following unilateral stroke in rats, agents that block Nogo or NgR enhance the growth of CST axon branches from the undamaged hemisphere into the denervated side of the spinal cord and improve use of the impaired forepaw. These effects can be seen using a recombinant soluble form of NgR, an anti-Nogo antibody, or by deleting the gene for NogoA/B or NgR (Papadopoulos et al., 2002; Lee et al., 2004; Cafferty and Strittmatter, 2006; Tsai et al., 2007). A recent study has shown that NEP1-40, the NgR antagonist used here, improves outcome after stroke, but did not investigate anatomical changes (Fang et al. 2010). NEP1-40 was previously shown to promote axon regeneration and improve outcome after spinal cord injury (GrandPre et al., 2002; Li and Strittmatter, 2003; Cao et al., 2008), though one group reported only weak effects in that model and found similar effects using a scrambled version of NEP1-40 (Steward et al., 2008). In the present study, NEP1-40 tripled the number of axon branches projecting from the intact CST to the denervated side of the spinal cord and improved use of the impaired forelimb. Preliminary studies from our lab show that a scrambled version of NEP1-40, while not enhancing axon growth over myelin in cell culture, may improve functional outcome after stroke. This effect may be related to an unanticipated resemblance between the scrambled peptide and a portion of MAG (Suppl. Fig. 3), enabling it to function in a fashion analogous to NEP1-40.

When combined with NEP1-40, inosine induced nearly twice as much CST sprouting on the denervated side of the spinal cord as either treatment alone, and enabled rats to retrieve food pellets with the impaired forepaw at the same level seen in normal animals. This high level of performance persisted for at least a month after treatment ended, suggesting that once formed, circuits that sustain behavioral improvements do not require continued application of plasticity-inducing agents. At the same time, the normal performance observed with the impaired paw suggests that the formation of new circuitry for ipsilateral motor control does not strongly compromise the ability of the intact hemisphere to control the contralateral paw. Although we have focused on the CST, in view of its role in the task used here (Raineteau et al., 2001; Anderson et al., 2005; Piecharka et al., 2005) and the relative ease in detecting newly formed ipsilateral projections, the exact relationship between the observed anatomical changes and the behavioral improvements is not known. Changes not investigated here may have also played a role in recovery, including possible novel inputs to the forelimb motor area of the undamaged hemisphere and to intact areas in the injured hemisphere (see, e.g., Li et al.; Carmichael et al., 2001; Frost et al., 2003; Dancause et al., 2005; Brown et al., 2008, 2009); descending pathways from spared areas of the injured hemisphere (Bareyre et al., 2004); and/or changes in the efficacy of existing synapses. It is clear that vicariation, the assumption of function by brain areas that did not previously mediate control of the impaired forepaw, occurs in rodent models of stroke even without further treatment (see Introduction), and inosine is likely to have enhanced this process. Establishing the significance of any brain changes to behavioral improvements may require lesions of candidate regions, transections of specific pathways, or agents that prevent the changes from occurring in the first place. We should also note that the present study quantified food retrieval as an endpoint but did not analyze the precise digit and limb movements underlying recovery. Other research in the field would suggest that the functional improvements commonly involve the use of compensatory strategies rather than recovery of the normal pattern of paw use (Whishaw et al., 1991). An additional point is that buprenorphine, which was used throughout as an analgesic, has anti-inflammatory effects (Volker et al., 2000) that may affect axonal sprouting in positive or negative ways (Yin et al., 2003; Steinmetz et al., 2005; Gensel et al., 2009; Stirling et al., 2009) and alter patterns of gene expression in the microarray studies.

The present results show that EE promotes CST axon sprouting from the intact side of the brain into the denervated side of the spinal cord after stroke and improves skilled reaching with the impaired forepaw. Inosine augmented the behavioral effects of EE, enabling rats to regain use of the impaired paw after a few weeks. The combination of the two treatments did not enhance CST reorganization beyond the level seen with inosine alone, but markedly altered gene expression in layer 5 pyramidal cells of the undamaged hemisphere. Compared to rats exposed to a standard environment, rats treated with inosine and exposed to EE showed changes in the expression of genes involved in cell signaling, cell morphology, cell growth, cell maintenance, assembly and organization; DNA replication, recombination and repair; and nervous system development and function. These observations may be linked to the previously noted effects of EE on chromatin remodeling (Fischer et al., 2007). That study and the present one suggest that, whereas rearing in isolation diminishes the overall level of neuronal gene transcription, EE, by fostering histone acetylation and DNA methylation, reverses this situation and enables inosine to influence gene transcription. From another perspective, the ability of EE to affect gene transcription was found to be augmented by inosine. The basis for this observation is unknown. However, our results suggest the possibility that chromatin remodeling and genome-wide transcriptional changes may depend on Mst3b activation, a hypothesis that will require further testing. It might be noted that the molecular changes associated with inosine in the present study do not overlap strongly with those observed previously (Zai et al., 2009), which may be due in part to differences in environmental rearing conditions in the two studies. Whereas rats in the prior study were housed two per cage, rats in the present microarray studies were either reared alone and offered almost no stimulation, or exposed to a great deal of stimulation (EE). Our results show that such differences can profoundly influence gene expression. At a behavioral level, combining NEP1-40 with training that includes many of the features of EE used in this study was recently shown to result in near-complete restoration of skilled motor performance with the impaired limb in mice (Fang et al., 2010). Finally, although the changes in gene expression correlate with behavioral improvements, we do not know which if any of the molecular changes enhances the ability of neurons to modify synaptic relationships in an adaptive fashion or promote axon branching. Further analysis of this relationship will require manipulations of candidate genes or their protein products.

Acknowledgments

We are grateful for the support of the NIH (R01 NS047446 to LB, NS39962 to SMS), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and Alseres Pharmaceuticals. We wish to thank the DDRC of Children’s Hospital (NIH P30 HD018655) for use of the Histology and Image Analysis Cores; Charles Vanderberg and the Laser Capture Core Facility of the Harvard Center for Neural Discovery; Jing Ou and Fuying Gao for assistance with microarray experiments; and Judith Li, Haleh Hashemi and Melissa Hootstein for animal training and testing.